Heterozygous Ala156Val Mutation in the GPIb Alpha (Heterozygous Bernard-Soulier Syndrome Type Bolzano) Induces Macrothrombocytopenia by Hampering Proplatelet Formation.

BACKGROUND. Pathogenesis of macrothrombocytopenia in Bernard-Soulier syndrome (BSS) is still obscure, although the normal amount of bone marrow megakaryocytes (MKs) and the normal platelet survival point towards defective platelet formation as the causative defect.

PATIENTS AND METHODS. We analyzed in vitro megakaryocyte differentiation and maturation, as well as proplatelet formation (PPF) in 6 patients heterozygous for the Ala156Val mutation in the GPIb alpha (Bolzano mutation). All of them had mild to moderate thrombocytopenia and enlarged platelets. MKs were differentiated from cord blood CD34⁺ cells (1 patient) and peripheral blood mononuclear cells (5 patients) for 12 days. Mature MKs were grown in suspension or plated onto glass coverslips coated with collagen I, fibrinogen (FNG), or von Willebrand Factor (VWF). MK differentiationmaturation and PPF were evaluated by phase contrast and fluorescence microscopy upon cell staining with anti alpha-tubulin and CD41 antibodies. Controls were analyzed in parallel with each patient sample.

RESULTS Immunomorphological evaluation of cultured cord blood progenitors at day 12 revealed that, the percentage of CD41+ cells with MK morphology was comparable in patient and relative control. Moreover, classification of MK morphology, according to standard criteria, revealed no significant differences in the maturation profiles between patient and control, indicating that the Bolzano mutation did not affect either differentiation or maturation of MKs. When reseeded in fresh medium, mature MKs derived from cord blood of both patient and control extended proplatelets. However, a time course analysis revealed a severe reduction of PPF by MKs derived from the patient: in fact, while 25% of control MKs were forming proplatelets after 48–72 hours of culture, the percentage of MKs with PPF from the patient’s cord blood never exceeded 5%. Adhesion to FNG or VWF resulted in a similar impairment of PPF with respect to control. As in the control, type I collagen totally inhibited PPF by BSS cord blood derived MKs. PPF by MKs derived from the peripheral blood of 5 BSS type Bolzano patients was also analyzed. As for the cord blood we observed a significant reduction of PPF, both when MKs were grown in suspension and when MKs were adhering to fibrinogen or VWF. Thus, as in MKs from cord blood, the Bolzano mutation seemed to affect PPF also in MKs cultured from peripheral blood. In morphologic analysis proplatelets derived from both cord and peripheral BSS type Bolzano blood revealed an altered structure of the tips that presented altered alpha-tubulin distribution and an enlarged diameter with respect to controls. Interestingly, staining of patients’ peripheral blood films, revealed that some of the circulating platelets had an altered alpha-tubulin distribution resembling that observed at proplatelets’ tips.

CONCLUSIONS. MKs from patients carrying the Bolzano mutation present both quantitative and qualitative abnormalities of in vitro PPF. This indicates a key role for GPIb in PPF and confirms the hypothesis that macrothrombocytopenia of BSS derives from defective platelet formation.